This invention relates to a method and paraphernalia for visually or photometrically detecting the presence or the absence of one or more microbial target analytes in a material sample; and enumerating the analyte(s) when found to be present in the sample. The material being analyzed can be a biological material; an environmental material; a food stuff material; or some other material which can harbor the target analyte(s). The target analyte(s) is (are) one or more specific living organisms such as microbes, bacteria, fungi, mycoplasma or protozoa which may be found in the material being tested; and the presence or absence of the target analyte or analytes is determined by causing metabolic reproduction of the target analyte or analytes in a suitable growth medium. The analysis can be performed in a gel or a semi-solid growth material which supports selective growth of the target analyte(s), but restricts growth of other viable organisms which may be in the sample. In this manner, detectable target analyte colonies are formed in the medium. The colonies may be detected and counted by various techniques, including the use of visually or photometrically detectable materials which will selectively bind to some portion of the target analyte(s).
The analysis of specimens for the presence or absence of target bacteria and other viable organisms such as fungi and protozoa, wherein the specimen is placed on a sterile growth medium, is well known. Various bacteria are usually differentiated by their appearance, their chemical reaction with growth or other medium, or by applying an enzymatic or immunological reactant. Since bacterial colonies of different species appear similar, it is usually necessary to perform a number of tests on a number of colonies before one can be establish a correct identification. This is especially a problem if it is necessary to distinguish bacterial subspecies, for example, finding the location of a colony of E. coli serotype 0157:H7 within a mix of other, apparently identical E. coli colonies. There are growth medium with specialized characteristics which help in the rapid visual location of such organisms, but the more closely related the target organism is to the background, the more difficult it is to distinguish them on chemical tests alone. It would be highly desirable if such colonies could be rapidly and specifically located, particularly if no additional operations were required by the technician.
This invention relates to a method and paraphernalia for detecting the presence or absence of one or more target microbial analytes in a sample. Detection of the target analyte is accomplished by growing the target analyte in a nutrient medium so as to form colonies of the target analyte in the medium, and then detecting and counting any resultant target analyte colonies which may form in the medium.
The analysis is performed in a medium which may also contain, or is combined with, one or more labeled analyte-specific materials (LASMs). The LASMs are homogeneously distributed throughout the medium The LASMs include a binding element, which may be a protein, lectin or a nucleotide sequence that is specific to a binding site in or on the target analyte. The label may be a dye or a fluorophore, including a colloid or particle, which is attached to the binding element. The LASMs in the medium can diffuse and migrate through the medium to the target analyte colonies, and will bind to the individual organisms in the colonies thereby differentially highlighting the colonies relative to the remainder of the medium. The LASMs may either bind directly to the surface of the organism, or to a product of the organism, such as its capsular material or an internal molecular structure, so long as the LASM, being so bound, does not appreciably migrate away from the site. Because the binding of the LASMs locally depletes the LASM concentration in the regions of the medium occupied by the target analyte colonies, a xe2x80x9chaloxe2x80x9d of lower LASM concentration will form around each target analyte colony, and at the center of each halo will be a high intensity peak corresponding to the labeled colony. Any non-target organisms present in the medium will not bind the LASMs and will show this characteristic.
A sample of a material to be analyzed is inoculated into the medium or distributed on the medium. If a quantitative analysis is desired, the amount of the sample inoculum will be volumetrically measured. As the organisms reproduce, colonies will form and will remain in a fixed location in or on the medium. The LASMs within the medium are sized so as to be able to migrate in or through the medium. Therefore, the LASMs in the region of the of the target analyte colonies are free to move through the medium and bind to the target organisms in the colonies, or to their products. This will cause the target organism colonies to show a higher label signal than surrounding areas in the medium. The reason for this is that the target organism colonies become LASM-enriched, and the surrounding areas become LASM-depleted. The immediumtely surrounding areas in the medium, which abut the target organism colonies, will tend to emit a weakened label signal. The net result is the formation of xe2x80x9cbrightxe2x80x9d spots surrounded by xe2x80x9cdimxe2x80x9d halos in the medium. In the event that there is no target analyte present in the sample, then the medium will retain a relatively even level of label signal emission, and will appear to be evenly xe2x80x9ccoloredxe2x80x9d.
The assay can be completed within one to two hours after inoculating the medium with the sample if a sensitive opto-mechanical photometric reader is used such as the one described in co-pending application U.S. Ser. No. 09/255,673, filed Feb. 23, 1999; or within about eighteen hours if a visual inspection is used. Since one will know the volume of the sample inoculated into or spread onto the medium, one can quantify the amount of the target analyte per unit volume of the specimen sample by counting the number of highlighted colonies In the medium. This enables the use of the invention to obtain a quantitative analysis for target analyte. It is desirable that a portion of the medium which contains the LASM be protected against sample inoculation, thus allowing this portion of the medium to serve as a negative control.
Some samples, especially those from xe2x80x9cdirtyxe2x80x9d sources, such as environmental samples, or those containing a great deal of other materials, may have a large variety of particles or other material that may have some level of non-specific signal, and where these non-specific signals may be confused with those from the target analytes. This problem can be overcome, however, by visually or photometrically taking sequential images from the same areas in the medium, which images are separated from each other by suitable periods of time. Only those colonies formed from the target organism will increase in signal intensity by virtue of their continued growth. A comparison of successive images, preferably performed by the use of digital image processing, can separate these areas of increasing intensity from the surrounding medium. This method of kinetic analysis is the preferred method in all circumstances where a suitable photometric reading device is available because this technique provides the greatest sensitivity, the best selectivity, and the fastest time to completion of the analysis.
In order to give the greatest signal/noise ratio, it is desirable that the medium be as thin as possible, i.e., about one millimeter or less, and preferably less than about 0.50 millimeter. The concentration of the label should be such that the signal from it can just be detected from a non-inoculated area of the medium. For a visual examination, a useful signal would be one that is slightly visible using the naked eye under the appropriate lighting conditions and is not so marked so as to obscure the signal from the colonies. For automated examination, the concentration should give an intensity which has an adequate signal-to-noise ratio over a xe2x80x9cdarkxe2x80x9d background which allows its quantitation. The exact amount must be determined based upon the photometric characteristics of the instrument used. Fluorophores such as fluorescein, sulforhodamine, and Cy-3, Cy-5 and Cy-7 can be used as labels with the photometric analysis protocol. Note that two or more target organisms can be detected using this method if the labels of the LASMs for each target can be distinguished, for example, by color, and/or light wave emissions from the LASMs.
It is therefore an object of this invention to provide a method and apparatus which can be used to assay a specimen sample for the presence or absence of a target analyte.
It is another object of this invention to provide a method and apparatus of the character described wherein the sample is placed on a medium which is combined with a target analyte-specific labeled material that can migrate within the medium.
It is a further object of this invention to provide a method and apparatus of the character described wherein the target analyte is a living organism.
It is another object of this invention to provide a method and apparatus of the character described which can detect two or more target analytes on the same medium.
It is an additional object of this invention to provide a method and apparatus of the character described wherein the target analyte is detected by growing colonies of the target analyte in the medium, and thus creating increasingly localized concentrations of the analyte-specific labeled material in the colonies of the target analyte.
These and other objects and advantages of the invention will become more readily apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings, in which: